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. 2026 Jul 12;74(9):e70197. doi: 10.1002/glia.70197

Genetic Deletion of Adenosine A2A Receptors Attenuates Aged‐Related Alterations of Glial Cells Morphology and of Inflammasome in the Hippocampus and Prefrontal Cortex of Mice

Cátia R Lopes 1,2, Samira G Ferreira 1,3, Rodrigo A Cunha 1,2,4, Paula Agostinho 1,2,4,✉
PMCID: PMC13358461  PMID: 42438359

ABSTRACT

Although brain disorders are the major burden of disease in Western countries and their incidence increases sharply with aging, the biological basis of brain aging is still poorly explored. Glial cells, namely microglia and astrocytes, maintain brain homeostasis and mount neuroinflammation that can contribute to age‐related deterioration of brain functions. The purinergic system, particularly adenosine A2A (A2AR) and P2X7 (P2X7R) receptors, modulates glial function and neuroinflammation. The present study aims to investigate how aging affects microglia and astrocytes morphology and the NRLP3 inflammasome complex, a key driver of the inflammatory process, and if the genetic deletion of A2AR has a protective role in inflammaging. We resorted to wild‐type and A2AR knockout mice with 3‐ and 24‐ month‐old to investigate alterations in microglia and astrocytes morphology, in P2X7R, and in related NRLP3 inflammasome components in the hippocampus and prefrontal cortex. Data show that brain aging alters the tridimensional structure of microglia and astrocytes in the hippocampus and prefrontal cortex. Aging decreased the levels of P2X7R and of inflammasome components, NLRP3 and caspase 1, in the hippocampus. Remarkably, A2AR knockout abrogated age‐related morphological changes of glial cells in both brain structures. Also, the decreased hippocampal P2X7R levels and the alterations in NLRP3 levels in both hippocampus and cortex, were no longer present in aged A2AR knockout mice. These findings indicate that A2AR might bolster NRLP3 inflammasome activation associated with an age‐related neuroinflammation, and A2AR blockade might promote healthy brain aging.

Keywords: A2A receptors, aging, astrocytes, brain, microglia, NRLP3 inflammasome, P2X7 receptors

  • Aging alters the tridimensional structure of microglia and astrocytes, and P2X7R and NLRP3 inflammasome levels.

  • Genetic deletion of A2AR protects age‐associated alterations of glial cell morphology and changes in P2X7R and NRLP3 levels.

graphic file with name GLIA-74-0-g002.jpg

Abbreviations

A2AR

adenosine A2A receptors

ASC

apoptosis‐associated speck‐like protein containing a caspase recruitment domain

CNS

central nervous system

GFAP

glial fibrillary acidic protein

IBA‐1

ionized calcium‐binding adaptor molecule 1

KO

knockout

NLRP3

NOD‐like receptor protein 3

PBS

phosphate buffered saline

PFA

paraformaldehyde

PFC

prefrontal cortex

RT

room temperature

WT

wild‐type

1. Introduction

Lifespan is increasing globally, and age is the primary risk factor for the development and progression of neurological disorders, thus making the understanding of age‐related brain changes a priority (Hou et al. 2019; World Health Organization 2024). Aging is characterized by an accumulation of senescent cells that produce a senescence‐associated secretory phenotype (SASP), which involves the increased release of growth factors and pro‐inflammatory cytokines, contributing to chronic, low‐grade inflammation—known as inflammaging—that can propagate CNS damage over time (Müller et al. 2025; Suk 2026). In fact, inflammaging contributes to dysfunction and ultimately leads to tissue damage that occurs upon aging (Franceschi and Campisi 2014; Ferrucci and Fabbri 2018; Schafer et al. 2020). Inflammatory responses in the brain are mostly ensured by the activity of glial cells, namely astrocytes and microglia, and their altered function upon aging is expected to be a major contributor to the dysregulation of neuroinflammatory responses in the aging brain (Semyanov et al. 2025).

We recently compiled a series of observations that supported a benefit on aging of the life‐long intake of moderate amounts of coffee (Lopes and Cunha 2024). The benefits of coffee on brain‐related behavior and diseases appear to mostly attributable to one of coffee's main constituents—caffeine (Grosso et al. 2017; Picó‐Pérez et al. 2023; Machado et al. 2024; Zhang et al. 2026). In nontoxic doses, caffeine mainly acts as an antagonist of adenosine receptors (Fredholm et al. 1999; Lopes et al. 2019), which operate a neuromodulation system bolstering the encoding of information salience in neuronal networks (reviewed in Cunha 2016). Accordingly, caffeine affords a robust neuroprotection in different animal models of brain diseases, an effect mimicked by selective antagonists of adenosine A2A receptors (A2AR) (Cunha 2016; Merighi et al. 2023). Notably, we recently reported that the genetic elimination of A2AR prevented alterations of synaptic plasticity and of memory in aged mice (Lopes et al. 2026). Although cortical A2AR are located in synapses (Rebola et al. 2005) and control synaptic plasticity (Rebola et al. 2008; Kerkhofs et al. 2018), A2AR are also present and control the function of both astrocytes, microglia and neuroinflammation (e.g., Orr et al. 2009; Rebola et al. 2011; Matos et al. 2013; Launay et al. 2025). Since glia cells are increasingly recognized to impact synaptic plasticity and behavior (Heneka et al. 2025; Meron Asher and Goshen 2025; Holt et al. 2026; Nakamura and Shichita 2026) and they are modified on aging (Verkhratsky et al. 2019; Suk 2026) and proposed to be triggers of age‐associated memory dysfunction (Lau et al. 2023; Rim et al. 2024; Hayashide et al. 2026), we now enquired if the genetic deletion of A2AR could prevent aging‐associated alterations of glia cells as well as triggers of neuroinflammation such as P2X7R and NLRP3 inflammasome (di Virgilio et al. 2023; Xu et al. 2025). We focused on the prefrontal cortex (PFC) and CA1 area of the hippocampus since these were the two brain areas where we previously found that A2AR controlled abnormal plasticity in aged mice.

2. Methods

2.1. Animals and Ethical Statements

Experiments were mainly conducted in wild‐type (WT) and A2AR global knockout (A2AR‐KO) mice with a C57BL/6 background (12 male and female mice of 3‐ and 24‐months old). Mice were housed with ad libitum access to food and water, under a controlled environment with 21°C ± 2°C and approximately 66% humidity and a fixed 12 h dark/light cycle. All studies were approved by the Institution's Ethical Committee (ORBEA_300/2021), conducted in agreement with the approved animal welfare guidelines and European legislation for the use of experimental animals and certified by Direção Geral de Alimentação e Veterinária (DGAV 018449 de 29‐11‐2021). All efforts were made to reduce the number of animals used and to minimize their stress and discomfort according to the guidelines of the European Union (Directive 2010/63/EU) and of the Portuguese law on animal care (113/2013). The sample size of mice used in this study was calculated based on our previous experience in neurochemical variability and range of expected effects (Madeira et al. 2023).

2.2. Immunohistochemistry

WT and A2AR‐KO mice were transcardially perfused with ice‐cold phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Brains were removed, postfixed for 24 h in PFA, dehydrated in 30% sucrose solution for 72 h at 4°C and cryopreserved at −80°C until use. Coronal brain sections (30 μm) were obtained, using a cryostat (CryoStar NX50, ThermoScientific, RRIDD:SCR_022732). For the immunolabeling protocol, free‐floating brain sections were washed with PBS, permeabilized in 0.1% Triton‐X100 solution in PBS, and nonspecific binding was blocked for 2 h with 10% horse serum and 0.1% Triton‐X100 solution in PBS. Then, sections were incubated overnight at 4°C, under agitation, with the primary antibodies: mouse antiglial fibrillary acidic protein (GFAP, 1:1000, Cell Signaling Technology Cat# 3670, RRID:AB_561049), rabbit anti‐ionized calcium‐binding adaptor molecule 1 (IBA‐1, 1:1000, FUJIFILM Wako Pure Chemical Corporation Cat# 019‐19741, RRID:AB_839504). Subsequently, slices were rinsed and incubated with the appropriate secondary antibodies: donkey anti‐mouse Alexa Fluor 488 (1:500, Thermo Fisher Scientific Cat# A‐21202, RRID:AB_141607), donkey anti‐rabbit Alexa Fluor 594 (1:500, Thermo Fisher Scientific Cat# A‐21207, RRID:AB_141637) for 2 h at room temperature (RT). After rinsing with PBS, slices were stained with the nuclear dye DAPI (1:5000) for 10 min at RT and washed with PBS before mounting the sections onto gelatin‐coated slides, using DAKO mounting medium. Sections were visualized using an epifluorescence microscopy (Zeiss, Axio Imager Z2 microscope, RRID:SCR_018856) and images were obtained with a Plan‐Apochromat ×10/0.45 objective. To carry out the tridimensional reconstruction of the microglial and astrocytic structure, Z‐stacks images were acquired with a step size of 0.45 μm along the z‐axis using a LSM 710 confocal inverted microscope (Zeiss, RRID:SCR_018063) with a Plan‐Apochromat ×40/1.2 DIC M27 objective.

2.3. Cell Counting

Counting of GFAP‐positive (GFAP+) and IBA‐1‐positive (IBA‐1+) cells (astrocytes and microglia, respectively) was done using the ROI manager in ImageJ, defining an area to measure the cell counting within: (a) stratum radiatum of CA1 hippocampal region and (b) prelimbic area of PFC. Data are shown as the percentage of immune‐positive cells per area compared to controls (3‐month‐old WT mice—mean of 100%).

2.4. Tridimensional Reconstruction of Microglia and Astrocytes

The morphology of astrocytes and of microglia was evaluated as previously described by us and others (Tavares et al. 2017; Madeira et al. 2023), keeping in mind that the thickness of the slices may truncate the morphology of glial cells, although this limitation affects all experimental groups. The tridimensional (3D) reconstruction of glial structures was performed in Z‐stack images using an open‐access tool, Simple Neurite Tracer (SNT, RRID:SCR_016566) plugin available in Fiji‐ImageJ software (RRID:SCR_002285). The 3D reconstruction of microglia and astrocytes processes was carried out within the stratum radiatum of the CA1 subregion of the hippocampus and prelimbic area of the PFC, which were selected based on our previous electrophysiological recordings (Lopes et al. 2026). Microglia and astrocytes were selected for the 3D reconstruction according to the following criteria: (i) a single nucleus enwrapped by an IBA‐1‐ or GFAP‐immunolabeled structure, (ii) the main structure of the microglia or astrocyte did not present truncated processes, and (iii) reconstruction was carried out in the first four microglia and astrocytes fulfilling the previously mentioned criteria in each animal of both ages and genotypes, being each animal considered an independent experiment. It should be highlighted that the PFC has a lower density of stained elements compared with the hippocampus and also has a different population(s) of GFAP‐immunoreactive astrocytes that may be differently stained with the low dilution of GFAP used; however, these limitations affected all experimental groups, allowing for comparisons to be made. The morphometric analysis of microglia and astrocytic arbor complexity was performed by quantifying the number of processes, and their total length, as well as by the Sholl analysis, which assesses the number of processes intersections using concentric spheres starting at the center of the soma with intervals of 4 or 2 μm, for astrocytes and microglia, respectively (York et al. 2018; Madeira et al. 2023).

2.5. Preparation of Total Protein Extracts

Total protein extracts were prepared as previously described (Lopes et al. 2023). Briefly, after animal euthanasia, the brains were dissected on an ice‐cold artificial cerebrospinal fluid (in mM: 124.0 NaCl, 4.4 KCl, 1.0 Na2HPO4, 25.0 NaHCO3, 2.0 CaCl2, 1.0 MgCl2, 10.0 glucose). The hippocampus and cerebral cortex were collected and stored at −20°C until use. Then these tissues were homogenized in radioimmunoprecipitation assay buffer (RIPA; Tris–HCl 50 mM pH 7.4, NaCl 150 mM, IGEPAL (NP‐40) 1%, sodium deoxycholate 0.5%, EDTA 1 mM and SDS 0.1%) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM dithiothreitol (DTT), 0.001% CLAP protease inhibitor cocktail (Sigma, USA), and PhosSTOPTM phosphate inhibitors (Roche, Switzerland). The homogenate was placed at 4°C with continuous agitation for 2 h, followed by centrifugation at 13,000g for 20 min at 4°C. The supernatant was collected and stored at −20°C until use. The determination of protein concentration was assessed with the BCA Protein Assay Kit (PierceTM, Thermo ScientificTM, USA).

2.6. Western Blotting

Western blot analysis was carried out as previously described (Lopes et al. 2023). Briefly, total protein extracts were resuspended in sample buffer 6× concentrated (500 mM Tris, 600 mM DTT, 10% SDS, 30% glycerol and 0.012% bromophenol) and heated at 70°C for denaturation for 20 min. After proteins electrophoresis and electro‐transfer to nitrocellulose membranes, a nonspecific block was performed by incubating the membrane with 5% milk or 3% bovine serum albumin (BSA) in Tris‐buffered saline (137 mM NaCl, 20 mM Tris, pH 7.6) containing 0.1% Tween 20 (TBS‐T) for 1 h at RT with agitation. The membranes were further incubated overnight at 4°C with the primary antibodies diluted in TBS‐T with 1% skim milk or 3% BSA, namely: rabbit antiapoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC/TMS1, 1:500, Proteintech Cat# 10500‐1‐AP, RRID:AB_2174862), rabbit anti‐NOD‐like receptor protein 3 (NLRP3, 1:1000, Cell Signaling Technology Cat# 13158, RRID:AB_2798134), rabbit anti‐caspase‐1/p20/p10 (1:1000, Proteintech Cat# 22915‐1‐AP, RRID: AB_2876874), rabbit anti‐P2X7 receptor (P2X7R, 1:500, Santa Cruz Biotechnology Cat# sc‐514962, RRID:AB_2892179). Then, membranes were washed with TBS‐T and incubated at RT with the secondary antibodies for 2 h. The secondary antibody used was a peroxidase‐conjugated anti‐rabbit IgG (1:10,000, Thermo Fischer Cat# 31462, RRID:AB_228338). Afterward, the membranes were washed with TBS‐T and revealed with an enhanced chemiluminescence (ECL) kit (PierceTM Thermo Scientific) or with Immobilon Forte (Millipore, USA). The immunobands were detected and analyzed using aChemiDoc equipment (BioRad, USA, RRID:SCR_019037) and further quantified using the ImageLab software. Then, membranes were re‐probed with an anti‐α‐tubulin antibody (1:20,000, Sigma‐Aldrich Cat# T6074, RRID:AB_477582) used to control the protein loading in the electrophoresis gel.

2.7. Statistical Analysis

Data were normally distributed, as verified using Shapiro–Wilk test. The ROUT test was used to identify outliers with Q = 1%. Data are presented as mean ± standard error of mean (SEM) of independent experiments, where each point represents a different animal of the different groups (Western blot analysis) or number (n) of cells counted or reconstructed (immunohistochemistry analysis). Parametric analysis was carried out after assessing the normal distribution of the groups. Unpaired Student's t‐test was used for comparisons between two different ages. For comparisons between different ages vs. genotypes, WT and A2AR‐KO mice with 3‐ and 24 months of age, we used a two‐way ANOVA followed by Tukey's post hoc test. Importantly, we reused the same data from WT mice with 3‐ and 24‐month‐old in these two analyses. In order to compare data obtained from Sholl analysis, we performed two‐way ANOVA followed by Sidak's multiple comparisons test to assess differences between the experimental groups along radial distance to the soma. Data of the % of GFAP+ and IBA‐1+ cells were expressed as percentages relative to control animals (WT mice with 3‐month‐old, 100%). Data obtained from Western blot experiments were expressed as differences relative to control animals (WT mice with 3 months ‐old used as internal control of each blot). Data are presented as mean ± SEM of the number of independent experiments (different animals). The within‐blot variability was controlled by a reprobing with α‐tubulin and the attenuation of the variability between blots was achieved by computing all immunoreactivity in comparison to a chosen control in each blot—the immunoreactivity of the sample collected from WT mice with 3‐month‐old. Since this internal normalization impeded the use of two‐way ANOVA analysis, we only computed the statistical significance of the differences found between each group relative to 3‐month‐old (control) using a sample t‐test comparing with the hypothetical value of 0 (control value). The significance level was set for p value < 0.05 in all tests. Statistical analyses were performed using GraphPad Prism Software version 8.1.1. (GraphPad Software, RRID:SCR_002798).

3. Results

Since (i) synaptic plasticity and excitatory/inhibitory balance is altered in the hippocampus and PFC (Lopes et al. 2026), (ii) astrocytes and microglia can control information flow in neuronal pathways (Heneka et al. 2025; Meron Asher and Goshen 2025; Holt et al. 2026; Nakamura and Shichita 2026), (iii) A2AR are upregulated in the aged limbic cortex of rodents and humans (e.g., Cunha et al. 1995; Canas et al. 2009; Temido‐Ferreira et al. 2020) and (iv) A2AR blockade attenuates the age‐related behavioral and electrophysiological alterations in the hippocampus and PFC (Lopes et al. 2026); we now characterized the age‐associated alterations of the morphology of microglia and of astrocytes in the hippocampus and PFC and tested if they were prevented upon genetic deletion of A2AR. Notably, we did not observed differences in the morphology of male and female mice within each experimental group, as also previously observed regarding age‐related behavior and electrophysiological properties (Lopes et al. 2026), as well as in adenosine neuromodulation (Valladão et al. 2025). Thus, the data presented below corresponds to the pooling of males and females.

3.1. Genetic Deletion of A2AR Avoids Age‐Related Alterations in Microglia Morphology

Microglia, the resident immune cells of the CNS, play essential roles in maintaining normal brain function; however, during aging, these cells may contribute to establish a hostile microenvironment to other brain cell types (Norden and Godbout 2013). In spite of their morpho‐functional complexity, the morphology of microglia can still be considered a simplified proxy of their pathophysiological role, with anti‐inflammatory and cell‐supporting microglia displaying greater ramification complexity whereas pro‐inflammatory ‘activated’ microglia display decreased complexity and a more spheroid shape (Paolicelli et al. 2022; Tremblay and Verkhratsky 2024). In the hippocampus, we observed no differences in the number of IBA‐1‐positive (IBA‐1+) cells in aged (24‐month‐old; 24 M) compared with young mice (3‐month‐old; 3 M), nor in A2AR‐KO of both ages (3 M A2AR‐KO: t11 = 1.49, p = 0.1645; 24 M WT: t 11 = 0.96, p = 0.3554; 24 M A2AR‐KO: t 11 = 0.9227, p = 0.3760, Figure 1b). However, regarding hippocampal microglia's 3D (see Figure 1a) structure of WT and A2AR‐KO mice with 3‐ and 24‐ month‐old, we observed that in aged mice (24‐month‐old) the microglia cells display a less complex morphology, with a significant reduction in the number of processes (p < 0.0001, Figure 1c) and in total processes length (p < 0.0014, Figure 1d) relatively to young adult (3‐month‐old) mice. Hence, Sholl analysis, which quantifies several indices of cellular morphology, revealed significant aging‐associated alterations in the 3D structure of hippocampal microglia (p < 0.0001, Figure 1e). In A2AR‐KO mice with 3‐month‐old, we did not observe alterations in microglia complexity compared with age‐matched WT mice, either in the number of processes (p = 0.4322, Turkey's post hoc test), total length (p = 0.9673; Turkey's post hoc test), or Sholl analysis (p < 0.0001). However, in aged A2AR‐KO mice (24‐month‐old), an increased number of processes (p = 0.0002, Turkey's post hoc test), total length (p = 0.007, Turkey's post hoc test), and Sholl analysis (p < 0.0001) were observed when compared with WT of the same age (Figure 1c–e).

FIGURE 1.

FIGURE 1

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Genetic deletion of A2AR avoided age‐related alterations in microglial morphology complexity in hippocampal and prefrontal cortex (PFC). Representative immunohistochemistry images showing IBA‐1 immunolabeling (red) and nuclei stained with DAPI (blue) in the CA1 region of hippocampus (a) and in the prelimbic PFC (f) of WT and A2AR‐KO mice with 3‐ and 24‐ month‐old. Scale bar: 25 μm. A2AR‐KO mice of both ages show no differences in the number of IBA‐1+ cells in either the hippocampus (b) or the PFC (g), expressed as a percentage of control (3‐month‐old WT mice = 100%). $$$ p < 0.001, one sample t‐test, hypothetical value of 100. Microglial morphology was evaluated by the number of processes (c, h), and total processes length (d, i), as well as by performing Sholl analysis (e, j), comparing microglia from WT and A2AR‐KO young and aged mice (3‐ and 24‐ month‐old, respectively). Results are mean ± SEM of 12 astrocytes (from 3 mice per group). *p < 0.05, **p < 0.01 using two‐way ANOVA post hoc Tukey's multiple comparisons test between indicated columns; ## p < 0.01, ### p < 0.001, #### p < 0.0001, compared to 3‐month‐old WT mice using a post hoc Tukey's test after two‐way ANOVA. Sholl analysis: ++ p < 0.01, +++ p < 0.001, two‐way ANOVA followed by Tukey's multiple comparison test (comparison between mice of 24‐month‐old).

Microglia 3D structure was also evaluated in PFC of WT and A2AR‐KO mice with 3‐ and 24‐months (Figure 1f). In aged WT mice (24‐month‐old), the 3D structure of microglia was different from WT mice with 3‐month‐old, being observed a significant decrease in microglia morphological complexity evaluated for the number of processes (Figure 1h) and for total processes length (Figure 1i) by Turkey's analysis after two‐way ANOVA (p < 0.0001). The impact of A2AR genetic deletion in 3‐month‐old mice was similar to that observed in the hippocampus: no differences in the number of processes (p = 0.998, Turkey's post hoc test) and total length (p = 0.980, Turkey's post hoc test), as well as in the values of Sholl analysis (p = 0.809) were observed between both groups (WT and A2AR‐KO mice Figure 1h–j). Though, the number of IBA‐1+ cells decreased in WT aged mice (24 M: t 11 = 4.84, p = 0.0005) but remained unaltered in A2AR‐KO mice of both ages (3 M A2AR‐KO: t 11 = 1.65, p = 0.1279; 24 M A2AR‐KO: t 11 = 0.70, p = 0.4991, Figure 1g). Overall, genetic deletion of A2AR avoided aging‐induced changes in microglial morphology, as A2AR‐KO mice with 24‐month‐old showed values comparable to age‐matched WT mice for the number of processes (p = 0.054, Tukey's post hoc test) and for their total length (p = 0.127). These findings indicate that, in both the hippocampus and PFC, A2AR genetic deletion mitigates age‐related alterations in microglial 3D structure.

3.2. Genetic Deletion of A2AR Avoids Age‐Related Alterations in Astrocytes Morphology

We next sought to detail age‐associated morphological alterations in astrocytes, immunolabeled for GFAP, and the impact thereupon of A2AR deletion in brain regions associated with cognitive decline, such as the hippocampus and the PFC (Figure 2a,f). In contrast to microglia, the morphology of astrocytes is not indicative of a particular pathophysiological role (Zhou et al. 2019; Baldwin et al. 2024), although alteration of astrocytic morphology is tightly coupled with altered functions, namely upon reactive astrogliosis (Pekny and Pekna 2014). Remarkably, aging did not affect the number of GFAP+ cells in the CA1 hippocampus of WT (control = 100%) and A2AR‐KO mice (3 M A2AR‐KO: t 11 = 0.95, p = 0.3622; 24 M WT: t 11 = 1.51, p = 0.1604; 24 M A2AR‐KO: t 11 = 0.54, p = 0.5985, Figure 2b). Although no changes were observed in the number of GFAP+ cells present in the hippocampus, there were some noticeable alterations in astrocyte morphology between the different mice groups. Data showed a significant increase in the number of processes (p < 0.0001, Turkey's post hoc test, Figure 2c) and in their total length (p < 0.0001, Turkey's post hoc test, Figure 2d) of aged WT mice compared to younger WT mice. In agreement with these findings, Sholl analysis revealed significant alterations in astrocytic 3D structure in aged hippocampus (p < 0.0001, Figure 2e). In 3‐month‐old mice, A2AR genetic deletion (A2AR‐KO) did not affect the 3D morphology of astrocytes, since no differences in the number of processes (p = 0.826, Turkey's post hoc test), in their total length (p = 0.998, Turkey's post hoc test), or in Sholl analysis values (p < 0.0001, Figure 2e) were observed, comparatively to 3‐month‐old WT mice. On the other hand, in 24‐month‐old mice, A2AR genetic deletion avoided the increase of astrocyte arborization complexity caused by aging, as can be observed from similar number of processes (p < 0.0001, Turkey's post hoc test) and their total length (p < 0.0001, Turkey's post hoc test), as well as from Sholl analysis values (p < 0.0001, Figure 2e) in aged A2AR‐KO mice compared to young (3‐month‐old) WT and A2AR‐KO mice.

FIGURE 2.

FIGURE 2

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Genetic deletion of A2AR avoided age‐related alterations in astrocyte morphology in the hippocampus and prefrontal cortex (PFC). (a) Representative immunohistochemistry images showing GFAP immunolabeling (green) and nuclei stained with DAPI (blue) in the CA1 region of the hippocampus of WT and A2AR‐KO mice with 3‐ and 24‐ month‐old and of prelimbic PFC (f). Scale bar: 25 μm. A2AR‐KO mice of both ages did not show differences in the number of GFAP+ cells in the hippocampus (b) but A2AR‐KO mice with 3‐month‐old showed a decrease in the number of these cells in the PFC (g), expressed as a percentage of control (3‐month‐old WT = 100%). $$ p < 0.01, one sample t‐test, hypothetical value of 100. Astrocytic morphology was evaluated by the number of (c, h), the total length (d, i), and also by Sholl analysis (e, j), comparing astrocytes from WT and A2AR‐KO young and aged mice (3‐ and 24‐ month old, respectively). Results are mean ± SEM of 12 astrocytes (for 3 mice per group). *p < 0.05, ***p < 0001 using two‐way ANOVA post hoc Tukey's multiple comparisons test between indicated columns; ## p < 0.01, #### p < 0.0001, compared to 3‐month‐old WT mice using a post hoc Tukey's test after two‐way ANOVA. Sholl analysis: ++ p < 0.01, two‐way ANOVA followed by Tukey's multiple comparison test (comparison between mice of 24‐month‐old).

In PFC, aging also did not alter the number of GFAP+ cells, but A2AR‐KO mice that were 3‐month‐old had significantly fewer GFAP+ cells as compared with 24 month‐old A2AR‐KO mice (3 M A2AR‐KO: t 11 = 3.46, p = 0.0053; 24 M WT: t 11 = 0.10, p = 0.9191; 24 M A2AR‐KO: t 11 = 1.44, p = 0.1783, Figure 2g). The observed changes in 3D structure of PFC astrocytes were qualitatively similar to those of the hippocampus, with an increase in aged mice in the number of processes (p = 0.0019, Figure 2h) and of their total length (p = 0.0004, Figure 2i). Also, Sholl analysis showed significant age‐related differences in astrocytic morphology in the PFC (p < 0.0001, Figure 2j). These data indicate that aging had a significant impact on astrocytic arborization complexity in the hippocampus and PFC. Nevertheless, at 3‐month‐old, A2AR‐KO mice did not show alterations in astrocytes' 3D configuration compared to WT mice (number of processes: p = 0.999, Turkey's post hoc test; total processes length: p = 0.781, Turkey's post hoc test; Sholl analysis: p = 0.1292, Figure 2h–j). Similarly to the hippocampus, at 24‐month‐old, A2AR‐KO mice displayed a significant decrease in arborization complexity compared with WT mice of the same age, as noticed by the reduced number of processes (p = 0.017, Turkey's post hoc test) and of their total (p = 0.035), and also by the values of Sholl analysis (p < 0.0001). Thus, A2AR genetic deletion avoided alterations in astrocyte morphology caused by aging in the hippocampus and in the PFC.

3.3. Impact of A2AR Genetic Deletion on the Levels of P2X7R and of Inflammasome Components in Aged Mice

P2X7R are ATP‐activated ion channels that are major triggers of neuroinflammation (di Virgilio et al. 2023) and activators of the inflammasome (Pelegrin 2021) in accordance with the role of ATP as a danger signal in the brain (Rodrigues et al. 2015). Hence, we evaluated the impact of aging on the levels of P2X7R and of inflammasome components in the hippocampus and cortex of WT and A2AR‐KO mice. The levels of these proteins were quantified by Western blot, using total extracts of the whole hippocampus and frontal cortex to have enough sample for analysis. Data show that there was a significant decrease in the levels of P2X7R in aged compared with younger WT mice (24 M WT: t 4 = 3.81, p = 0.0189, Figure 3a). On the contrary, in A2AR‐KO mice no differences (t 6 = 0.08, p = 0.9372) in the levels of P2X7R were observed when compared to WT mice of 3‐month‐old (control set at 0%). Notably, the decrease of P2X7R levels observed in WT mice with 24‐month‐old was not observed in aged A2AR‐KO mice (24 M A2AR‐KO: t 5 = 0.90, p = 0.4085; Figure 3a).

FIGURE 3.

FIGURE 3

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Genetic deletion of A2AR normalized the levels of P2X7R in the hippocampus and cortex of aged mice. Variation of the density of P2X7R, expressed as a percentage relative to control animals (3‐month‐old WT mice, 0%, dashed line), in the hippocampus (a) and in the cortex (b) of WT and A2AR‐KO mice. Representative immunoreactive bands are shown on the top of bar graphs, corresponding to P2X7R and to protein loading control (α‐tubulin) evaluated in 3‐ and 24‐month‐old WT and A2AR‐KO mice. Data are mean ± SEM of n = 5–8 independent experiments, with each point corresponding to an individual animal of each group. # p < 0.05, one sample t‐test, hypothetical value of 0.

On the other hand, aged mice did not show differences in P2X7R levels in the cortex (24 M WT: t 6 = 0.35, p = 0.7402, Figure 3b), as compared with 3‐month‐old WT mice. Also, no significant changes in P2X7R levels in A2AR‐KO mice of both ages (comparing to 3 M WT; 3 M A2AR‐KO: t 7 = 0.72, p = 0.4976; 24 M A2AR‐KO: t 5 = 0.08, p = 0.9356; Figure 3b) were observed. Overall, these results indicate that the age‐related changes in P2X7R levels are no longer observed in the hippocampus of aged mice with a genetic deletion of A2AR.

There is evidence linking the activation of P2X7R and the assembly of NRLP3 inflammasome, an intracellular multiprotein complex triggering neuroinflammation, that is involved in the aging process (reviewed in Liang et al. 2024). Thus, we evaluated the levels of the inflammasome complex, NRLP3, ASC and caspase 1, in the hippocampus and cortex in adult and aged WT and A2AR‐KO mice (Figure 4). In the hippocampus of 24‐month‐old WT mice, NLRP3 (24 M WT: t 2 = 5.75, p = 0.0289, Figure 4a) and caspase 1 levels (24 M WT: t 6 = 3.88, p = 0.0082, Figure 4c) were decreased, without any significant difference in ASC levels (24 M WT: t 6 = 1.45, p = 0.1986, Figure 4b). Regarding age‐related alteration in inflammasome components of A2AR‐KO mice, it was observed that in young (3‐month‐old) A2AR‐KO mice, the levels of NLRP3 were significantly lower (3 M A2AR‐KO: t 3 = 3.20, p = 0.0494), although aged A2AR‐KO mice displayed NLRP3 levels (24 M A2AR‐KO: t 5 = 0.96, p = 0.3823; Figure 4a) similar to control levels (3‐month‐old WT) in hippocampal total extracts. On the other hand, caspase 1 levels in young A2AR‐KO mice remained unaltered, compared to age‐matched WT mice (t 3 = 1.28, p = 0.2901 vs. 3 M WT), and the alterations of caspase 1 in aged mice were maintained in mice with genetic deletion of A2AR (compared to 3 M WT; 24 M A2AR‐KO: t 4 = 5.31, p = 0.0061; Figure 4c). In the cortex, we observed a significant increase in NLRP3 (24 M WT: t 4 = 2.96, p = 0.0415; Figure 4d) levels in aged mice and no difference either in ASC (24 M WT: t 8 = 1.38, p = 0.2048; Figure 4e) or in caspase 1 (24 M WT: t 8 = 0.80, p = 0.4447, Figure 4f). However, no significant differences in NLRP3 levels were observed between WT and A2AR‐KO mice with 3‐month‐old (3 M A2AR‐KO: t 6 = 1.89, p = 0.1077); in aged A2AR‐KO mice the NLRP3 levels were not significant different (24 M A2AR‐KO: t 5 = 1.47, p = 0.2024; Figure 4d) to those found in controls mice, that is, 3‐month‐old WT mice. These results indicate that the absence of A2AR in aged mice abrogated the changes in NLRP3 levels caused by aging.

FIGURE 4.

FIGURE 4

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Age‐associated alterations in the NLRP3 levels in the hippocampus and cortex of aged mice were no longer observed in A2AR knockout mice. The density of inflammasome components, expressed as a percentage relative to control animals (3‐month‐old WT mice, 0%, dashed line), in the hippocampus: NLRP3 (a), ASC (b), and caspase 1 (c), and in the cortex: NLRP3 (d), ASC (e), caspase 1 (f). Representative immunoreactive bands are shown on the top of bar graphs, corresponding to proteins of interest and to the protein loading control (α‐tubulin) evaluated in 3‐ and 24‐month‐old WT and A2AR‐KO mice. Data are mean ± SEM of n = 3–7 mice per group. # p < 0.05, ## p < 0.01, one sample t‐test versus the control value of 0.

4. Discussion

The present study characterized alterations in the morphology of microglia and astrocytes in the hippocampus and PFC of aged mice. In aged mice, microglia exhibited atrophy with fewer and shorter processes, whereas astrocytes displayed an increase in ramifications and in their length. These modifications were accompanied by altered levels of NLRP3 inflammasome, mainly in its sensor, coactivator, and effector proteins (NLRP3, P2X7R, and caspase 1, respectively) in aged mice. Remarkably, in mice with genetic deletion of A2AR (A2AR‐KO mice), no age‐related alterations were observed neither in the morphology of microglia and astrocytes nor in the levels of P2X7R and NLRP3 in the hippocampus and cortex.

Our results show that aging significantly altered the morphology of microglia and astrocytes, in line with results described by others (Jinno 2011; Fabricius et al. 2013; Robillard et al. 2016). In the case of microglia, morphological complexity decreased in aged mice, typified by a reduction in the number and in the length of their processes in the hippocampus and PFC, as well as a reduction in the number of microglia (IBA‐1+ cells) in the PFC. Age‐related alterations in microglia involve a shift from a resting or surveying state, exhibiting a small soma highly branched in thin processes, to a more ‘activated’ morphology, marked by hypertrophy with enlarged soma and retracted, thicker, and less complex processes, or to a dystrophic or senescent phenotype, usually coupled with structural fragmentation and functional decline (Ogura et al. 1994; Streit et al. 2004; Davies et al. 2017; reviewed in Wendimu and Hooks 2022); this might be associated with a chronic low‐grade microglia activation characteristic of an inflammaging process (Franceschi et al. 2018; Kempuraj et al. 2026).

In contrast to the changes observed in microglia, age‐related alterations in astrocytes morphology consisted of an increased complexity of astrocytic processes, exhibiting an augmented arborization, that is, greater complexity; however, aging did not significantly affect the number of astrocytes (GFAP+ cells) in the hippocampus and PFC. It is worth noting that changes in the morphology of astrocytes with aging are not consensual. Some previous studies reported an increased size and complexity of astrocytic processes (Jinno 2011; Grosche et al. 2013; Bondi et al. 2021), whereas other studies reported an atrophy of astrocytes in aged human brain with shorter and thinner terminal processes or branches (Jyothi et al. 2015; Palmer and Ousman 2018; Popov et al. 2023) and in aged primates (Kanaan et al. 2010; Robillard et al. 2016) and rodents (Amenta et al. 1998; Popov et al. 2021). These differences may result from different methodologies used to assess astrocytes morphology, and from the heterogeneity in astrocytes morphology and size across different species and their potential reactive states (Oberheim et al. 2012; Zhou et al. 2019). Currently, no available tools allow to accurately characterize the complete astrocytic morphology, particularly of the ultra‐thin processes (Baldwin et al. 2024). We are aware of the limitations of GFAP immunolabeling, widely used to visualize astrocytes, but all experimental animal groups in this study were analyzed using the same methodology; therefore, the differences found in aged astrocytes suggest that these cells might be mediating a compensatory response to restore brain alterations upon aging, which we recently showed to involve changes in synaptic plasticity in the hippocampus and PFC (Lopes et al. 2026).

Age‐related changes in the morphology of glial cells can impact the physical and functional interactions between astrocytes, microglia, and the pre‐ and postsynaptic compartments, forming the quadripartite synapse (Schafer et al. 2013; Garland et al. 2022), which can affect synaptic plasticity and memory both in the hippocampus and PFC, as reported by others (Tanaka et al. 2013; Popov et al. 2021; Lawal et al. 2022). Previous findings correlated astrocyte morphology with their capacity to enwrap synapses (Pannasch et al. 2014), which changes the physical and metabolic support of neurons (reviewed in Ghézali et al. 2016). Accordingly, our group reported a parallel alteration of astrocyte morphology and of synaptic plasticity in young adult mice (Pereira et al. 2021). Furthermore, microglia during aging lose their capability to remodel myelin, impacting normal neuronal function (Luan et al. 2021). It is also known that aberrant synaptic pruning, which is mediated mainly by glial cells, is associated with neurodegeneration and cognitive deficits (Gomez‐Arboledas et al. 2021; Guedes et al. 2022); thus, it would be interesting to further investigate the potential relationship between age‐related glial cells modifications, particularly in complement proteins, and morpho‐functional changes of synapses. Given the morphological and functional diversity of glial cells, supporting the existence of distinct astrocyte and microglial populations (Matias et al. 2019; Tan et al. 2020; Delage et al. 2021), age‐related alterations in glial cells may be region‐specific (Rodríguez et al. 2014; Wendimu and Hooks 2022; Man et al. 2024) and may predict the selective vulnerability of brain regions during aging, which also need to be further explored.

Brain aging is associated with a chronic low‐grade inflammatory state, often referred to as neuroinflammaging, which is partly driven by microglia and astrocytes. This state is prevalent in aging and age‐related disorders, with inflammasome activation being a key component of this inflammaging (Franceschi et al. 2018; Brahadeeswaran et al. 2022). We also now investigated if aging impacted NLRP3 inflammasome and P2X7R activated by ATP, a danger signal leading to neuroinflammation (Rodrigues et al. 2015; di Virgilio et al. 2023). We provide the first evidence of a decrease in P2X7R levels in the hippocampus of aged mice, as also shown in an aged mouse model of retinal degeneration (Franke et al. 2005). We also observed a decrease in NLRP3 and caspase 1 levels in the hippocampus, in contrast with another study (Wang et al. 2022), and an increase in NLRP3 levels in the cortex, as reported in the senescence accelerated mouse‐8 (SAMP8) aging model (Xu et al. 2020). This decrease in hippocampal P2X7R density, closely correlating with the reduced levels of NLRP3 and caspase 1 and accompanied by decreased NLRP3 and caspase 1 levels, reveals an unexpected biological paradox. In the context of neuroinflammaging, enhanced activation of the P2X7R/NLRP3 inflammasome axis would be anticipated, especially given the pronounced morphological alterations observed in aged glia cells. Possible explanations for this apparent discrepancy may reside in a compensatory decrease of triggers of an already increased neuroinflammation or may reflect a progressive age‐related dysfunction in the astrocyte‐microglia communication, leading to a dissociation between morphological glial reactivity and canonical inflammasome signaling. Curiously, it was shown that genetic deletion of NLRP3 has different effects in different brain regions (Youm et al. 2013). Likewise, we and others also observed opposite neurophysiological alterations in the cerebral cortex and hippocampus of aged animals (Stanley et al. 2012; Lee et al. 2015; Lopes et al. 2026), suggesting that some of these changes might be compensatory mechanisms.

Remarkably, we showed that A2AR‐KO mice did not display the age‐related alterations in both astrocytes and microglia. In the hippocampus, A2AR elimination prevented the morphological age‐related alterations of astrocytes and microglia, without affecting the number of glial cells, whereas in the PFC, A2AR elimination eliminated the age‐related changes in the number of processes, but not the total process length, of astrocytes and microglia. These findings are in line with data from our previous study showing that aged A2AR‐KO mice also failed to display cognitive alteration and changes of synaptic plasticity in the hippocampus and PFC (Lopes et al. 2026). Adenosine receptors in distinct cell types exert opposing effects on brain function (Shen et al. 2008, 2013), possibly due to a spatiotemporally controlled activation of A2AR in different brain regions (Chen et al. 2023). This may explain the different effects of A2AR elimination on microglial and astrocytic morphology in the hippocampus and PFC. These results prompt the possibility that A2AR may be involved in the modulation of the neuroinflammatory process through the control of microglia and astrocytes morphologies, in accordance with previous studies showing the capacity of A2AR to control cell remodeling (Ribeiro et al. 2016; Xu et al. 2022), namely of microglia (Caetano et al. 2017; Simões‐Henriques et al. 2020) and astrocytes (Brambilla et al. 2003). We previously showed that A2AR antagonism attenuates the neuroinflammation‐induced activation and morphological changes of microglial cells induced by the systemic administration of lipopolysaccharide (LPS), an endotoxin from gram‐negative bacteria (Rebola et al. 2011). Notably, we now showed that A2AR can control alteration of the levels of NLRP3 inflammasome in the aged brain. Importantly, A2AR activation does not activate but rather potentiates inflammasome activity through a cAMP/PKA/CREB/HIF‐1α pathway in cultured mouse peritoneal macrophages (Ouyang et al. 2013). The present study also shows that the genetic deletion of A2AR avoids the age‐related changes in the levels of P2X7R, a major trigger of neuroinflammation (Rodrigues et al. 2015; di Virgilio et al. 2023), in agreement with our previous findings showing a functional interaction between A2AR and P2X7R in controlling neuroinflammatory processes in rats subjected to chronic stress (Dias et al. 2021), as well as in cultured astrocytes (Dias et al. 2022). This provides novel evidence for an interaction between these two arms of the purinergic system (reviewed in Agostinho et al. 2020) now in the control of age‐related brain dysfunction. However, the molecular mechanisms underlying this interaction remain to be clarified. There are some possibilities to explain this interaction, such as (i) formation of P2X7R and A2AR heteromers (Antonio et al. 2011; Ferré and Ciruela 2019); (ii) the signaling pathways activated by one receptor can modulate the function of the other receptor (Garção et al. 2013; Miras‐Portugal et al. 2019; Temido‐Ferreira et al. 2020); (iii) balanced activation of both receptors due to ATP catabolism into adenosine by ecto‐nucleotidases (Dunwiddie et al. 1997; Cunha et al. 1998; Cunha 2001; Kukley et al. 2004).

In conclusion, the present study shows that aging alters astrocytic and microglial morphology and changes the density of P2X7R, NLRP3 and caspase 1 in the hippocampus and in the cortex, mainly in PFC, two brain regions involved in cognitive function (reviewed in Jobson et al. 2021). Importantly, we now provide the first evidence that the genetic deletion of A2AR abrogated most of the age‐related changes of the morphology of microglia and astrocytes. Our data also suggests that the A2AR play a key role in NRLP3 inflammasome activation during aging, through a pathway involving the P2X7R. These findings highlight the need to investigate in detail the mechanisms underlying the A2AR and P2X7R interaction and how this impact on a neuroinflammatory response, involving NLRP3 inflammasome activity, to fully explore the therapeutic potential of A2AR and, or P2X7R in controlling inflammasome activation in age‐related inflammatory process. This is of particular interest since deregulated and/or primed glia contribute to neurodegeneration, synaptic and cognitive deficits, and increased susceptibility to age‐related disorders, such as Alzheimer's disease (see Rohden et al. 2025).

Author Contributions

C.R.L., R.A.C., and P.A. conceived and designed the study. C.R.L. conducted the neurochemical studies. C.R.L. and S.G.F. performed image acquisition and tridimensional reconstructions. R.A.C. and P.A. provided the funding. C.R.L., R.A.C., and P.A. wrote the manuscript. All authors reviewed and approved the paper.

Funding

This work was supported by Programa Operacional Regional do Centro, Centro 2020 (CENTRO‐01‐0246‐FEDER‐000010), FCT (COMPETE2030‐FEDER‐0067630 and UIDB/04539/2020), and European Union's Horizon 2020 research and innovation program (Grant 857524). C.R.L. was supported by a fellowship from FCT—Fundação para a Ciência e Tecnologia, reference 2021.06954.BD, and DOI identifier https://doi.org/10.54499/2021.06954.BD.

Ethics Statement

All studies were approved by the Institution's Ethical Committee (ORBEA_300/2021) and certified by Direção Geral de Alimentação e Veterinária (018449, 29‐11‐2021) and conducted in agreement with European legislation (Directive 2010/63/EU) and the Portuguese law on animal care (113/2013).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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